Cross-sectional evaluation of the saccharin transit time test for primary ciliary dyskinesia: did we discard this tool too soon?

ABSTRACT BACKGROUND: Primary ciliary dyskinesia (PCD) is a rare and heterogeneous disease that is difficult to diagnose and requires complex and expensive diagnostic tools. The saccharin transit time test is a simple and inexpensive tool that may assist in screening patients with PCD. OBJECTIVES: This study aimed to compare changes in the electron microscopy findings with clinical variables and saccharin tests in individuals diagnosed with clinical PCD (cPCD) and a control group. DESIGN AND SETTING: An observational cross-sectional study was conducted in an otorhinolaryngology outpatient clinic from August 2012 to April 2021. METHOD: Patients with cPCD underwent clinical screening questionnaires, nasal endoscopy, the saccharin transit time test, and nasal biopsy for transmission electron microscopy. RESULTS: Thirty-four patients with cPCD were evaluated. The most prevalent clinical comorbidities in the cPCD group were recurrent pneumonia, bronchiectasis, and chronic rhinosinusitis. Electron microscopy confirmed the clinical diagnosis of PCD in 16 of the 34 (47.1%) patients. CONCLUSION: The saccharin test could assist in screening patients with PCD due to its association with clinical alterations related to PCD.


INTRODUCTION
Primary ciliary dyskinesia (PCD) is an autosomal recessive disease in which ciliary motility is compromised by mucus accumulation, changes in the microbiota of the airways, infection, structural changes with consequent functional worsening, and important clinical repercussions. 1,2 The clinical changes secondary to ciliary motility dysfunction include defects in laterality (situs inversus, situs inversus totalis, and dextrocardia), infertility, chronic rhinosinusitis (CRS), chronic otitis media, and recurrent infections of the upper and lower airways. [1][2][3] In the lungs, changes in mucociliary clearance are related to respiratory failure in the neonatal period, recurrent pneumonia, bronchiectasis, and chronic cough. 1,[3][4][5] There is no gold standard for PCD diagnosis. 1,2 It is a rare disease that affects approximately 1:10,000 live births. 1 Furthermore, it is a constantly evolving condition in terms of diagnosis, and some PCD phenotypes are not yet fully established. 1,2 Currently, guidelines from the American Thoracic Society (ATS) and the European Respiratory Society (ERS) suggest diagnostic confirmation through a combination of different clinical suspicion and diagnostic methods such as nasal nitric oxide (nNO), transmission electron microscopy (TEM), high-speed video microscopy, and genetic screening for pathogenic variants in PCDrelated genes. 1,6 Clinical scores such as the Primary Ciliary Dyskinesia Rule (PICADAR) and ATS clinical screening questionnaires can help in diagnosing this disorder. 1,6,7 TEM involves the analysis of ciliated epithelium samples. In this analysis, alterations in the ciliary ultrastructure were evaluated, and approximately 70% of patients with PCD presented with alterations within the TEM. 8 Some patients with PCD may not present obvious defects under TEM, even with changes in ciliary function. 2,8 Infectious and inflammatory processes can affect mucociliary transport, so false positives can be found in these cases. 9  The saccharin test allows for a rough evaluation of mucociliary function. It is a screening test that is widely available outside specialized centers; it is simple, inexpensive, easy to implement, and can be a useful tool for general practitioners. 10 Patients with a clinical PCD (cPCD) diagnosis based on otorhinolaryngology, pneumopediatrics, and pulmonology between August 2012 and April 2021 were included in the cPCD group.
The clinical diagnosis was based on the characteristic symptoms described by the ERS task force criteria: defects of laterality, family history of PCD, persistent rhinorrhea, CRS, neonatal respiratory failure, productive cough, bronchiectasis, chronic otitis (chronic otitis media, serous otitis media, conductive hearing loss), and infertility. 1 Patients diagnosed with cystic fibrosis, alpha-1-antitrypsin deficiency, or immunodeficiencies, and smokers were excluded.
Patients with insufficient material for TEM were excluded. Patients who presented with acute upper airway infections on the day of the appointment were rescheduled.
Because PCD is a rare disease, the sample size was based on a convenience sample of patients who agreed to participate in the study.
All patients answered a clinical form containing demographic data, characteristic PCD symptoms, and personal history, and those evaluated after 2016 answered the PICADAR and ATS clinical screening questionnaires. 6,7 All patients underwent nasal endoscopy, and the main findings were documented. This examination allowed the nasal fossa to be biopsied and tested, excluding obstructive factors.
The saccharin test was performed as described previously. 10,11,15 A sodic saccharin fragment measuring 1 mm in diameter was placed on the surface of the head of the inferior nasal turbinate 1 cm posterior to the nasal vestibule to avoid the squamous epithelium area. The participants remained seated, breathing normally, without sneezing or blowing their nose. The time between the placement of saccharin and the beginning of the sensation of sweet taste was measured in minutes. If the patient did not report a sensation of taste after 60 minutes, the test was interrupted. The test was considered altered when the result was greater than 30 minutes. 11 For the TEM evaluation, the material was collected through cytological brushing of the inferior turbinate. The material was placed in as container with a glutaraldehyde fixing solution of 3% and kept at 4 °C for three hours. The biopsy specimens were processed, washed, and placed in a phosphate buffer container. The samples were analyzed by two researchers (MDCT and EO) according to the international consensus guideline for reporting TEM (BEAT PCD TEM criteria). 16 Changes in the ultrastructure were based on the observation of at least 100 cilia, being evaluated in cross-sections. 1 Abnormalities found in less than 10% of the cilia were considered within the normal range. 17 Described alterations associated with PCD were analyzed, such as the absence of the internal and external arm of dynein, translocations, and absences of central microtubules, compound cilia, ciliary disorientation, and alterations of peripheral and central microtubules. 18,19 The BEAT PCD TEM criteria consist of class  The median saccharin progression time was 11.5 minutes. No association was found between PICADAR ≥ 7 or the ATS clinical score ≥ 2 and saccharin test greater than 30 minutes. Also, there was no association between PICADAR ≥ 7 or the ATS clinical score ≥ 2 and TEM class I defects.
* Eleven participants with a clinical diagnosis of primary ciliary dyskinesia (cPCD) were excluded: eight due to insufficient material for TEM and three for positive genetic testing for cystic fibrosis. ** 34 patients were included in the cPCD group; however, it was not possible to perform the saccharin test in four cases.

DISCUSSION
The diagnosis of PCD remains a major challenge in clinical practice due to the need for a combination of tools, which often require sophisticated techniques available in only a few centers in a limited number of countries. 13 The investigation of this disease becomes even more difficult due to the low incidence and great variability of genotypes and phenotypes. 7 In Brazil, few studies have been conducted that evaluate the diagnosis and clinical characteristics of patients with PCD. 20 Due to this phenotypic unpredictability, great heterogeneity may be observed in the clinical characteristics of these patients.
In this study, clinical variability was observed in the cPCD group, and the most frequent features were recurrent pneumonia, bronchiectasis, and CRS.
Identifying children with suspected PCD at an early age can improve the prognosis and delay pulmonary remodeling, leading to a decrease in pulmonary function. 6 years old for other conditions such as adenoid hypertrophy and even healthy children. [27][28][29] Our study found no association between altered clinical scores, such as the PICADAR and ATS-CSQ, and alterations in the saccharin test or TEM. Clinical scores have gained great relevance in the diagnostic algorithm, especially in the ATS guidelines, where patients with a clinical screening questionnaire score of less than two should not continue the investigation. 2 The positive predictive value of these scores in previous studies was similar to that of nNO, but these scores require multicentric and multidisciplinary validation. 13 The complete diagnostic algorithm for PCD can cost €653 to €2,097 per patient, which can be challenging in countries with limited resources and social heterogeneity, such as Brazil. This is not only due to the costs but also the lack of reference centers with staff able to perform the required tests and analyses. 26,30 Thus, physicians should pay attention to patients with severe or atypical symptoms and individually evaluate each patient's clinical history. 1 In this context, the assessment of the saccharin transit time may be an additional tool to corroborate subjective clinical decisions, particularly in primary and secondary care centers.
Our study has some limitations because it examined a rare disease and reduced the number of patients evaluated per year. In the nine years of analysis included in this study, there were changes in the diagnostic criteria, especially concerning the TEM criteria, and scores such as the PICADAR and ATS clinical questionnaire were incorporated before 2016. In addition, evaluating other tools is challenging because of the lack of a reference test for PCD diagnosis. Access to nNO, ciliary beat analysis through video microscopy, and genetic testing may be useful in future studies that diagnostic and screening tools.